Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa

ARTICLE Received 6 Apr 2015 | Accepted 20 Oct 2015 | Published 24 Nov 2015 DOI: 10.1038/ncomms9952 OPEN Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa G. Borgonie1,2, B. Linage-Alvarez2,*, A.O. Ojo2,*, S.O.C Mundle3,*, L.B. Freese4,*, C. Van Rooyen2, O. Kuloyo2,w, J. Albertyn2, C. Pohl2, E.D. Cason2, J. Vermeulen2, C. Pienaar4, D. Litthauer2, H. Van Niekerk5, J. Van Eeden5, B. Sherwood Lollar3, T.C. Onstott6 & E. Van Heerden2 Following the discovery of the first Eukarya in the deep subsurface, intense interest has developed to understand the diversity of eukaryotes living in these extreme environments. We identified that Platyhelminthes, Rotifera, Annelida and Arthropoda are thriving at 1.4 km depths in palaeometeoric fissure water up to 12,300 yr old in South African mines. Protozoa and Fungi have also been identified; however, they are present in low numbers. Characterization of the different species reveals that many are opportunistic organisms with an origin due to recharge from surface waters rather than soil leaching. This is the first known study to demonstrate the in situ distribution of biofilms on fissure rock faces using video documentation. Calculations suggest that food, not dissolved oxygen is the limiting factor for eukaryal population growth. The discovery of a group of Eukarya underground has important implications for the search for life on other planets in our solar system. 1 Extreme Life Isyensya (ELI), PB 65, 9050 Gentbrugge, Belgium. 2 Department of Microbial, Biochemical and Food Biotechnology, Swot Street 9300 Bloemfontein, Republic of South Africa. 3 Department of Earth Sciences, University of Toronto, 22 Russell Street, Toronto, Ontario, Canada M5S 3B1. 4 AngloGold Ashanti Kopanang Mine, Private Bag X5010, Vaal Reef, North West 2621, Republic of South Africa. 5 Sibanye Gold Ltd, Driefontein Operations, Farm Leeupoort P111, Goudveld 2507, Republic of South Africa. 6 Department of Geosciences, Princeton University, B79 Guyot Hall, Princeton, 08544 New Jersey, USA. * These authors contributed equally to this work. w Present address: Department of Geoscience, University of Calgary, 2500 University Drive Northwest Calgary, Alberta, Canada T2N 1N4. Correspondence and requests for materials should be addressed to G.B. (email: ). NATURE COMMUNICATIONS | 6:8952 | DOI: 10.1038/ncomms9952 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9952 T he discovery of nematodes thriving in fracture and fissure water of South African gold mines1, which were neither too extreme in temperature nor too depleted in O2 concentration, raised the question as to why only nematodes were found. Several other species of lower invertebrates should have been able to thrive in these extreme environments but were not previously discovered. An extensive 2-year continuous sampling campaign was executed in deep-subsurface South African gold mines, which focused on capturing other Eukarya from flowing fracture waters using modified trapping filters. For the first time, video technology was used to capture footage of the unique biosphere environment accessed via mine boreholes, where Eukarya reside inside the fluid. This was extended to other mines where camera equipment could be inserted deep enough into a borehole. Chemical and microbial analysis of the fissure water, 3H and 14C concentration measurements in combination with extensive control sampling around boreholes and service water established the indigenous nature of the specimens recovered. We report on the discovery in deep-subsurface fissure biofilm of Protozoa, Fungi, Platyhelminthes, Rotifera, Annelida, Arthropoda and additional Nematoda. Calculations indicate that food availability, not O2, is a limiting factor for population growth. Video footage shows several types of biofilms growing on the fissure rock face, and collection of that biofilm establishes that this is the site where the Eukarya reside. The discovery of a complex group of interacting Eukarya in the deep subsurface indicates the biosphere on Earth is larger than previously determined and is significant for the search for life on other planets, particularly the planet Mars. Results Sample collection. To determine whether the Eukarya recovered were indigenous to the deep subsurface or resulted from recent surface sourced and/or from mining activities, the following steps were taken. Filtration procedures that had been successful in previous studies to collect nematodes from high volumes of borehole water (41,000 l) were repeated1; control samples were collected and tested for Eukarya from puddles/soil near the sampled boreholes and from the service water used in mining operation; the chemical composition (Supplementary Table 1) and the structure of the microbial community in the fracture water were determined; and 3H and 14C concentrations were measured to estimate the age and potential influence of meteoric recharge on the borehole water. Water samples were collected at 1.0 and 1.4 km depths from two boreholes in two different South African gold mines (Kopanang and Driefontein) (Table 1). A total of 654,821 l of fissure water was filtered from an open borehole at Kopanang over a 5-month period. A total of 12,845,647 l of fissure water from a valved borehole at Driefontein over a 2-year period. Both sampled boreholes were drilled using air rather than water, thereby eliminating any potential for contamination from service water. To characterize background levels and establish baseline conditions that could have contributed to contaminating the borehole water, three soil/ puddle samples were collected and two samples of service water were collected at each mine. A 50,400-l sample was collected at Kopanang using an inline filter and a 3,865,654-l sample was collected from Driefontein using a eukaryal trapping filter. The use of inline filters is only suitable for shorter periods and is not reliable over 2 years of continuous sampling. In both cases, total DNA was extracted and was negative for Eukarya. The control filter at Driefontein was attached as long as the sampling was done; in Kopanang, the control filter was prematurely removed 2 due to maintenance work that is periodically executed at the cooling plant. DNA extraction of bacterial filters and control filter. Genomic DNA isolated from all layers of the Kopanang filter yielded 600 ng for the fissure water filter and 80 ng for the service water filter (the control). In both cases, the internal and external net layers yielded the highest concentrations of DNA relative to the internal and external filter layers (Supplementary Fig. 1A,B). Gel electrophoresis of the service water (control) filter indicated that the fragment size ranged from 200 to 3,000 base pairs (bp), suggesting that the genomic DNA is highly degraded. PCR amplification of the 16S and 18S rRNA gene for the service water (control) DN (...truncated)